The name polybiguanide (PBG) is used to describe a diverse group of polymers containing repeating biguanide groups and may be linear or branched. In addition the biguanide groups may part of the main chain or incorporated as side-groups or end-groups in the compound. PBGs are easily and inexpensively synthesized in large quantities and the reaction yields stable products with several potential reactive sites for further modifications. PBGs are made by the condensation polymerization of a biscyanoguanidine and a diamino compound. Typically, the final PBG product has amino and monocyanoguanidine end groups at opposite ends of a cationic polymer backbone (see FIG. 1). These end groups can be subsequently reacted with either monofunctional amino- or cyanoguanidine-containing moieties, respectively (the transferred terminal moieties are termed end-caps). Also, attaching endcaps to the polybiguanide chain can be made with other reactive groups as is known in organic chemistry. In FIG. 1, “X” is the hydrocarbon segment between the biguanide group introduced from the biscyanoguanidine monomer and “Y” is the hydrocarbon segment introduced from the diamino co-monomer. The PBG polymer (FIG. 1) lends itself to modifications and/or additions, namely: (i) size and nature of backbone segment “X,” (ii) size and nature of backbone “Y,” (iii) terminal amino end-cap, (iv) terminal cyanoguanidine end-cap, and (v) anions. The synthesized product possesses a distribution of molecular weights that are readily separated by HPLC, or other means, to distinct fractions with definite molecular formulae. PBGs are fully ionized at physiologic pH. Other synthesis routs may be used to produce the polybiguanide compounds by one skilled the art of organic and polymer chemistry.
Previous polybiguanides have been described in the prior art, therein there are no modified end-groups, mono end cap modification and di end cap modification (to the best of our knowledge). Representative prior art patents comprise UK patents 702,268; 1,152,243; 1,167,249; 1,432,345; and U.S. Pat. Nos. 4,403,078; 4,558,159; 4,891,423; 5,741,886; and patent application number 2003/0032768 A1.
Advantages of PBGs as HIV-1 Microbicides
PBGs are already known for their wide-spectrum anti-bacterial activity and safety, e.g. as a contact lens disinfectant for over thirty years (Woodcock P. M. Biguanides as Industrial Biocides. In: K. R. Payne (ed), Critical Reports on Applied Chemistry: Industrial Biocides, vol. 23 John Wiley and Sons, New York). Because of their special biological functions, these well-established low cellular toxicity compounds have potential as STD microbicides as well as conventional antiviral agents. The following features represent important technical and economic advantages of PBGs that have been noted to date: (i) high activity against a wide range of organisms even in the presence of organic matter, (ii) low mammalian toxicity (Bratt and Hathway 1976 Macromolecular Chemistry 177:2591-2605; Jangaard et al. 1968 Diabetes 17:96-104; Czyzyk et al. 1968 Diabetes 17:492-498; ICI Bulletin Cosmocil CQ—an antimicrobial agent for use in cosmetics and pharmaceuticals. ICI Americas, Inc.), (iii) absence of odor, (iv) easy handling and application, (v) chemical stability and non-volatility, (vi) no surface activity; PBGs are not surfactants, i.e. they do not lower the surface tension of water or dissolve cellular membranes like surfactants, (vii) inexpensive, (viii) easy to prepare, and (ix) greater than 96% non-metabolized (Bratt and Hathway 1976 Macromolecular Chemistry 177:2591-2605; Jangaard et al. 1968 Diabetes 17:96-104; Czyzyk et al. 1968 Diabetes 17:492-498). The starting chemicals needed to make PBGs are commercially available in large quantities and their large-scale production is straightforward. Because of the exorbitant expense of antiviral therapy in the developing world, the concept of low cost antiviral agents to prevent the transmission or to combat existing infections has emerged as one of the most paramount of needs in the world today. Effective antiviral agents having different mechanism(s) of action and low cost or low cost microbicides would be a highly desirable addition to existing therapies, especially where female control of STD would help dramatically decrease transmission.
Safety of Biguanides and PBGs:
Extensive toxicological studies, covering different exposures to tissue targets and pathways, have demonstrated the safety of PBGs (Bratt and Hathway 1976 Macromolecular Chemistry 177:2591-2605; Jangaard et al. 1968 Diabetes 17:96-104; Czyzyk et al. 1968 Diabetes 17:492-498). Notably, chlorhexidine gluconate (CHG), a bis-biguanide, has been used as a general disinfectant for over thirty years with a high level of safety (Rabe and Hillier 2000 Sex Transm Dis 27:74-78; Shubair et al. 1992 Gynecol Obstet Invest 34:229-233; Stray-Pederson et al. 1999 Int. J. Antimicrob Agents 12:245-251). Many reports support the safety of CHG in gynecology and obstetrics as a vaginal douche or as a pre-delivery vaginal wash (Shubair et al. 1992 Gynecol Obstet Invest 34:229-233; Stray-Pederson et al. 1999 Int. J. Antimicrob Agents 12:245-251). For example, Rabe and Hillier report the use of 0.25% chlorhexidine gel is safe when used vaginally against chlamydia (Rabe and Hillier 2000 Sex Transm Dis 27:74-78; Patton et al. 1998 Sexually Transmitted Diseases 25:421-426). With vaginal use, CHG did not disturb flora with respect to Lactobacilli species (Shubair et al. 1992 Gynecol Obstet Invest 34:229-233). Polymeric PBGs have shown less corneal toxicity, compared to CHG, especially in contact lens applications (Woodcock P. M. Biguanides as Industrial Biocides. In: K. R. Payne (ed), Critical Reports on Applied Chemistry: Industrial Biocides, vol. 23 John Wiley and Sons, New York). Further, biguanide-based drugs have excellent safety profiles as an anti-malaria drugs (Proguanil) (Leggat and Haydon 2002 J. Travel Med. 9:156-159; Chaulet et al. 2002 Arzeimittelforschung 52:407-412; Croft and Herxheimer 2002 Clin Infect Dis. discussion: 1278-1279) and for treating type 2 diabetes (Metformin) (Stepensky et al. 2002 Drug Metab. Dispos 30:861-868; Zuhri-Yafi et al. 2002 J. Pediatri Endocrinol Metab 15 Suppl 1:541-546; Wulffele et al. 2002 Br J. Clin Pharmacol 53:549 P-550P; Melikian et al. 2002 Clin Ther 24:460-467) with a daily dose of 2.5 gm. In addition Cazzanig et al. (2002 Wounds 14:169-176) and Davis et al. (2002 Wounds 14:252-256), report that polyhexamethylene biguanide can form a barrier to prevent Pseudomonas wound invasion, while Ansorg et al. (Chemotherapy 48:129-133) have tested the biguanide poly hexanide against Staphylococcus aureus in the nasal mucosa. Welk et al. (J. Clin. Periodontology 29:392-399) used PHMB, which has been used as an antiseptic for many years, as a mouth rinse at 0.12% and found it significantly more effective in inhibiting plaque than placebo. Therefore, the positively charged biguanide class of compounds have consistently demonstrated safety profiles that allowed for their use in human studies up to and including regulatory approvals.
Virus Inactivation mechanisms of PBGs: PBGs in general are multi-action compounds that could interfere with virus infection by interaction at the cellular or viral membrane. Their accepted biological function, as antibacterial agents, is attributed to their interaction with cell membranes, specifically anionic phospholipids and possibly proteins (Woodcock P. M. Biguanides as Industrial Biocides. In: K. R. Payne (ed), Critical Reports on Applied Chemistry: Industrial Biocides, vol. 23 John Wiley and Sons, New York; Gilbert et al. 1990 J. Appl Bacteriol 69:585-592; Broxton et al. 1984 J. Applied Bacteriol 57:115-124; Broxton et al. 1983 J. Appl Bacteriol 54:345-353; Gilbert et al. 1990 J. Appl Bacteriol 69:593-598; Broxton et al. 1984 Microbios 41:15-22).
FIG. 2 depicts the multi-level functions of PBGs as potential antiviral microbicides. First, due to their cationic nature, PBGs could retard the movement of virions by binding to their negatively charged surfaces before they reach the cell surfaces. Second, PBGs could inhibit cell-free and cell-associated virus by interacting with viral envelope lipids or negatively charged viral proteins or with low affinity cell surface receptors in a non-specific fashion. Third, PBGs could effect cross-linking of sialic acid groups of mucin and increase its viscosity and ability to function as a physical barrier to prevent infective agents from reaching the epithelium. Fourth, PBGs could bind to acidic phospholipids causing changes in lipid and protein distribution in the cell and viral membranes with the result of inhibiting viral infectivity, possibly due to dislocation or conformational changes in viral receptors or co-receptors or due to inhibiting the fusion step. Fifth, PBGs could bind specifically to high affinity virus receptors on the cell surface and therefore inhibit virus attachment, and/or fusion.
To further illustrate this last point we will present data in the examples section of this patent application that shows PBG compounds that have specific interactions with the HIV-1 co-receptors CXCR4 and CCR5. These data taken together with the recent reports of positively charged peptides binding to CXCR4 and inhibiting T cell line tropic strains of HIV (De Clercq, E. 2002 New Anti-HIV Agents and Targets. Medicinal Research Reviews 22:531-565), suggests that a similar mechanism of action is part of the polybiguanide spectrum of antiviral activity. The chemical nature of PBGs with variation in the length of the backbone linkers (X and Y in FIG. 1A) may allow for the formation of a defined three-dimensional structure that together with the positive charge characteristics of the PBG class of molecules could lead to a defined, specific mechanism of action such as that observed for the positively charged peptides (De Clercq, E. 2002 New Anti-HIV Agents and Targets. Medicinal Research Reviews 22:531-565). In addition, B. V. Shetty has disclosed a series of guanidine or biguanide compounds with antiviral and antimicrobial activity (Shetty Application Published under the PCT, International Publication Number WO 02/17916 A1, Mar. 7, 2002). It is apparent from the work of Shetty and others that positively charged compounds can be developed as antiviral agents with specific molecular targets.
Persistence—importance of PBGs binding ability: Due to their cationic nature, selected PBGs are expected to strongly interact with both free virus and cell surfaces due to the strong electrostatic interaction between PBGs and anionic phospholipid groups. We predict that these strong electrostatic interactions will ensure that dilution or washing do not readily reverse PBG binding in the vaginal environment. To minimize the effect of the cationic charge of PBGs on the overall cellular sensitivity, several strategies have been identified, including: modulating the charge density by inserting special moieties in the backbone chain, altering the pKa of the biguanide group (e.g., by substituting it with amidine, pKa=9.5 or guanidine, pKa=13.0), tailoring the chain length or end-caps and selecting their chemistry and optimizing the anion conjugated with the PBG cation. The ability to design PBGs with vastly different physical characteristics led to the identification of PEHMB (PBG in which X=2 carbon atoms and Y=6 carbon atoms in FIG. 1A) which is more potent and less toxic than all others in this class of compounds tested to date. The reason for this may lie in the nature of the three dimensional structure of PEHMB which we believe imparts on the molecule a degree of specificity in its mechanism of action. Our preliminary data indicates that of all the PBGs tested at least PEHMB interacts with cellular receptors in a specific fashion therefore, we can theorize, this specificity imparts on PEHMB a superior antiviral profile and reduced cellular toxicity with respect to other members of this class of molecule.
The present invention relates to compositions and methods for inhibiting the transmission of enveloped viruses such as alphavirus, herpes viruses (e.g. HSV-1 to HSV-8, cytomegalovirus, varicella zoster, Epstein Bar Virus, etc.), rhabdoviruses, orthomyxoviruses (e.g. influenza), retroviruses (e.g. human immunodeficiency virus type 1, HIV-1), flaviviridae (e.g. Hepatitis C, West Nile, Dengue, and yellow fever viruses), and Pox viruses (e.g. smallpox, and vaccinia viruses).
Human immunodeficiency virus type 1 (HIV-1), a member of the retrovirus family, is the causative agent in the development of acquired immune deficiency syndrome (AIDS). This condition is a catastrophic, fatal disease that presently infects millions of people worldwide. Major efforts are being made to develop novel antiviral agents with unique mechanisms of action to be used in drug therapy and on methods of preventing the transmission of HIV-1, methods of curing the AIDS disease state once contracted, and methods of ameliorating the symptoms of AIDS.
Despite almost 20 years of AIDS/HIV-1 prevention efforts and research, the sexually transmitted HIV-1 epidemic continues to be a major health problem throughout the world and is accelerating in many areas. To date the HIV epidemic has infected over 42 million people predominantly through sexual intercourse at the end of 2002. Of these there has been 3.1 cumulative deaths from the disease worldwide (from the Joint United Nations Program on HIV/AIDS and the World Health Organization's AIDS Epidemic Update Report, December 2002).
Virtually all the compounds that are currently used or are the subject of advanced clinical trials for the treatment of HIV infections belong to one of the following classes:
1) Nucleoside analogue inhibitors of reverse transcriptase functions.
2) Non-nucleoside analogue inhibitors of reverse transcriptase functions
3) HIV-1 Protease inhibitors.
4) Virus fusion inhibitors (the 36 amino acid fusion inhibitor T20 has been approved for sale by the FDA)
The HIV-1 replication cycle can be interrupted at many different points. As indicated by the approved medications the viral reverse transcriptase and protease enzymes are good molecular targets as is the entire process by which the virus fuses to and injects itself into host cells. Thus the recently approved drug T20 is the first in a novel class of anti-HIV-1 agents. However, in addition to the drugs already approved for treatment of HIV-1 infection, work continues on the discovery and development of additional treatment modalities because of the virus's propensity to mutant and thus renders ineffective the existing therapies.
At present combination therapy comprising at least three anti-HIV drugs has become the standard treatment for HIV infected patients. Virtually all drugs that have been licensed for clinical use for the treatment of HIV infection fall into one of the four categories listed above, comprising three molecular targets. However one problem with current therapy is the cost associated with the need to use multiple drugs used in combination. Estimates of $15000 to $20000 U.S. per year per person are close approximations. This cost makes it virtually impossible for many people to afford combination therapy, especially in developing nations where the need is greatest. Another problem with existing therapeutic regimens, as stated above, is the ability of the virus to develop resistance to the individual medications and many times to develop resistance to the combination therapy. This works against the population in two ways. First, the individual infected will eventually run out of treatment options and second, if the infected individual passes along a virus already resistant to many existing therapeutic agents, the newly infected individual will have a more limited treatment option than the first. Therefore, the need for new, improved and hopefully inexpensive medications is evident.
Most importantly in the search for new medications to combat the spread of the HIV-1 virus is the search for chemotherapeutic interventions that work by novel mechanism(s) of action. Several potential areas for intervention that are under consideration or have active programs in include 1) blocking the viral envelope glycoprotein gp120, 2) additional mechanisms beyond gp120 to block virus entry such as blocking the virus receptor CD4 or co-receptors CXCR4 or CCR5, 3) viral assembly and disassembly through targeting the zinc finder domain of the viral nucleo capsid protein 7 (NCp7) and 4) by interfering with the functions of the viral integrase protein, and by interruption of virus specific transcription processes.
Vaginal contraceptive products have been available for many years and usually contain nonoxynol-9 or other detergent/surfactant as the active ingredient that are toxic to cell membranes. However frequent use of N-9 causes irritation and inflammation of the vagina (M. K. Stafford et al. J. AIDS human retrovirology, 1998 17:327-331). N-9 is also toxic to vaginal and cervical cells increasing the permeability of vaginal tissue, and can inactivate lactobacilli. Lactobacilli produce lactic acid and hydrogen peroxide that serve to maintain the acidic pH of the vagina (˜pH 3.5 to 5.0). At this pH, a number of sexually transmitted disease (STD) causing organisms like HIV, and spermatozoa are inactivated. Disturbance of the vaginal microbial flora can lead to vaginal infections, which in turn increase the chance of HIV/STD transmission. The most recent data (Stephenson, J. Am. Assoc. 2000, page 284-949) in which a topical formulated N-9 product strongly suggest that the compound may even enhance HIV transmission.
Therefore it is extremely important to identify and evaluate new contraceptive antimicrobial agents, microbicides, which can be used vaginally in effective doses without inactivating lactobacilli or causing overt vaginal irritation or other toxicity.
A successful microbicide should (i) be effective against infection caused by cell-free and cell-associated virus, (ii) adsorbs tightly with its molecular target(s), i.e., its adsorption should not be reversed by dilution or washing, (iii) permanently “inactivate” the virus, (iv) inactivate free virus and infected cells faster than their rate of transport through the mucus layer, (v) have persistent activity for more than one episode of coitus, (vi) be safe to host cells and tissues—causing no irritation or lesions, (vii) be easy to formulate, (viii) remain stable in the formulated state, (ix) not activate mucosal immunity, (x) retard transport in mucus and entire vaginal and rectal mucosa, and (xi) be inexpensive for worldwide application.
Current HIV-1 microbicide candidates fall into two categories—either surfactants or polyanionic compounds (Pauwels and De Clercq 1996 J. AIDS Hum Retroviruses 11:211-221; Recommendations for the development of vaginal microbicides. 1996 International Working Group on Vaginal Microbicies AIDS 10:1-6). However, these proposed agents may not satisfy all of the necessary criteria for a successful microbicide as mentioned above. In addition, most of the compounds under current investigations as microbicides are non-specific and emerged from either excipients or related compounds used in conventional topical formulations—almost none of the compounds used have definite chemical formulae, and many are based on natural or synthetic water-soluble polymers. For example, despite the effectiveness of N-9 with respect to HIV-1 inactivation in vitro, its failure to effectively prevent HIV-1 infection in vivo has been attributed to its high irritation profile and indiscriminate disruption of epithelial cells (Feldblum et al. 1986 N.C. Med J. 47:569-572; Alexander, 1990 WHO Global Programme on AIDS Fertil Steril. 54:1-18; Niruthisard et al. 1991 Sex Transm Dis 18:176-179; Roddy et al. 1993 J STD AIDS 4:165-170; Kreiss et al. JAMA 1992 268:477-482). In order to satisfy the diverse criteria stated above, the target molecule needs to be custom tailored to provide several functions at the same time. The ability to manipulate by synthetic means the molecular structure of the current classes of agents under investigation as potential microbicides (such as N-9 or C31G surfactants, or sulfated polysaccharides) is limited, or in some cases even impossible. In contrast polybiguanide-based molecules provide a wealth of possibilities with respect to targeted synthetic manipulation. These compounds are safe, inexpensive, and highly effective anti-HIV-1 microbicides that can be synthesized with or without spermicidal activity.
Herpes viruses are another class of virus that like HIV-1 develop resistance to existing therapy, and can cause problems from a STD (especially Herpes simplex virus type 2, HSV2) as well as a chronic infection point of view. For example human cytomegalovirus (HCMV) is a serious, life threatening opportunistic pathogen in immuno compromised individuals such as AIDS patients (Macher et al. 1983 NEJM 309:1454; Tyms et al. 1989 J Anitmicrob Chemother 23:89-105) or organ transplant recipients (Meyers, J. D., 1991 Am J. Med. 81:27-38). Over the past decade there has been a tremendous effort dedicated to improving the available treatments for herpes viruses. At the present time acyclovir is still the most prescribed drug for HSV1 and HSV2, while for HCMV ganciclovir, foscarnet, cidofovir, and fomivirsen are the only drugs currently available (Bedard et al. 2000 Antimicrobial Agents and Chemotherapy 44:929-937). None of the current treatments for herpesviruses are effective at preventing the sexual transmission of the viruses therefore there is still an urgent need for new drugs that have unique mechanisms of action and modes of therapeutic intervention.